The neurotoxins produced by Clostridium botulinum (Botulinum Neurotoxins, BoNT) are among the most poisonous substances known. The neurotoxin type E (BoNT/E) forms part of a family of seven related serotypes (botulinum toxins A to G) produced by different strains of Clostridium botulinum. The major types of human diseases caused by these toxins include food-borne botulism, infant botulism, wound botulism, and adult interstinal colonization. But these toxins can also constitute a potential biological weapon to the extent that they are easy to produce. On the other hand, for several years, the botulinum toxins, have been used for therapeutic (dystonia, neuronal hyperactivity such as strabismus, blepharospasm etc.) or aesthetic applications (in particular wrinkle reduction). For all these applications, it is essential to have a simple, quick and sensitive method for the detection and quantification of botulinum toxin in various media, including biological media.
The botulinum neurotoxins are synthesized by C. botulinum as inactive single chain polypeptides of 150 kDa which undergo proteolytic cleavage to generate active holotoxins constituting of two protein sub-units: a heavy chain (100 kD) linked to a light chain (50 kD) via a disulfide bridge. The heavy chain is involved in the binding of the toxin to the nerve ending, in the internalization then in the translocation of the light chain into the cytosol. The light chain is responsible for the toxicity of the protein by inhibition of the calcium-dependent release of acetylcholine. Unlike some types of botulinum neurotoxins (e.g. type A) that are generated as active dichain form, BoNT/E is released from the bacterium in an inactive form that must be activated by exposing to exogenous proteases such as trypsin.
The toxicity of the light chain of these toxins is due to its peptidase activity. In fact, the botulinum toxins belong to the family of zinc metallopeptidases and more particularly to the sub-family of the zincins which contain the consensus sequence HExxH; (Schiavo et al. (1992) J. Biol. Chem. 267, 23479-23483; Roques B. P. (1993) Biochem. Soc. Trans. 21, 678-685). They cleave very specifically the neuronal proteins involved in the exocytosis of the neurotransmitters, such as SNAP-25 and synaptobrevin. The cleavage site is specific to each toxin, including to an identical substrate. Botulinum toxin type E cleaves specifically one of the SNARE complex proteins, SNAP-25 (Chen et al., J Biol Chem. 2006 Apr. 21; 281(16): 10906-11, incorporated by reference in its entirety herein).
There are several challenges in the diagnosis and treatment of botulism. Since the toxicity of BoNT is so great, it is necessary to detect BoNT at very low concentrations, preferably as the active toxin. It is also imperative that the diagnosis be made rapidly since the equine-based treatment can have several substantial side effects.
The current standard for detecting botulinum toxins in a sample is the determination of the median lethal dose (LD50) in mice (Kautter and Salomon (1976) J. Assoc. Anal. Chem., 60, 541-545; incorporated by reference in its entirety herein). The mouse bioassay is currently the gold standard and is the only widely accepted method for the detection of BoNT. Mixtures of neutralizing antibodies are given to mice in conjunction with the sample in question to differentiate the toxin serotype. Mice receiving the appropriate anti-BoNT serotype antibody along with the toxic sample do not show symptoms and survive, while mice treated with the other serotype antibodies show symptoms and die. Importantly, this assay measures only active toxin. The mouse bioassay is very sensitive, detecting as little as 10 picograms (pg) of active toxin which is defined as 1 mouse LD50 (mLD50) or 1 unit of BoNT. However, the mouse bioassay can be slow (taking up to 4 days) for final results and it requires the sacrifice of many animals. It is highly desirable to have a more rapid technique of detecting botulinum toxins.
The ELISA is much more rapid, but is less sensitive, is problematic in certain matrices, shows cross reactivity between BoNT serotypes, and measures inactive toxin along with active toxin. The ELISA is currently used primarily as a fast screening technique and results are verified by the mouse bioassay.
Accordingly, it is an object of the present invention to provide such a sensitive detection and differentiation method for detecting botulinum neurotoxins using novel peptide substrates.